1. Field of the Invention
The present invention relates to immobilized enzymes and particularly relates to immobilized glucose isomerase and methods of producing same.
2. The Prior Art
The term `glucose isomerase` is the generic name used for those enzymes which transform glucose to fructose, and their main application lies in the production of fructose from glucose. That is, glucose isomerases are currently employed industrially for the production of fructose-containing syrups by isomerization of glucose. This reaction has conventionally been carried out by the batch method, with a solution containing a high concentration of glucose being contacted with a glucose isomerase for about 48 hours at a temperature of 60.degree. to 70.degree. C. However, since this reaction is performed in batches, there have been problems such as a poor utilization rate of the glucose isomerase, discoloration of the product due to the reaction being carried out for extended periods at high temperatures, high costs for refining the product following the reaction, and so on.
In addition, in recent years industrial applications of continuous isomerization methods have been developed using immobilized glucose isomerases prepared by adsorbing or binding a glucose isomerase on a special carriers, for example, an ion exchange resin or DEAE-cellulose.
Glucose isomerases are generally produced inside the cells of microorganisms. That is to say, most of the produced glucose isomerase exists inside the cell wall or on the cell wall of the producing microorganism. For this reason, in order to carry out adsorption of a glucose isomerase on a carrier such as an ion exchange resin, the glucose isomerase must first be separated from the cells of the microorganism and employed in the form of a solution. Some examples of methods employing glucose isomerase in this way in the form of a solution are disclosed in U.S. Pat. Nos. 3,708,397; 3,788,945; 3,850,751 and 3,868,304. However, when following these methods for achieving adsorption of glucose isomerase on a carrier such as an ion exchange resin, there are various problems encountered such as a low level of adsorption of the glucose isomerase on the ion exchange resin or other carrier, and this results in the obtained immobilized glucose isomerase having a low isomerizing efficiency in the continuous isomerization system.
It has now been found, in accordance with the present invention, that a polysaccharide which is contained in the abovementioned solutions of glucose isomerase is competitively or preferentially adsorbed on the ion exchange resin or other carrier, and this adsorption of polysaccharide acts to inhibit the adsorption of the glucose isomerase itself on the ion exchange resin or other kind of carrier. That is, it was found that an adsorbed polysaccharide makes it difficult to immobilize glucose isomerases on carriers such as ion exchange resins. For this reason the glucose isomerase adsorbed on the ion exchange resin or other carrier is low. It should be understood in this context that "polysaccharides" means higher molecular weight saccharides which are not released into the dialysate and are retained in the glucose isomerase solution when said glucose isomerase solution is dialysed for one night against deionized water containing 10 mM MgCl.sub.2 and 1 mM CoCl.sub.2.
Further, in accordance with the present invention, it has been discovered that it is possible to adsorb a large amount of glucose isomerase on a carrier such as an ion exchange resin employed for the immobilization of said glucose isomerase if said contaminating polysaccharide is first eliminated from the cultured material obtained by culturing a microorganism producing glucose isomerase. It has also been found that it is possible to achieve an elevated efficiency in the isomerization reaction when the immobilized glucose isomerase obtained in this way is employed in a continuous isomerization system. Based on this finding, the present inventor obtained U.S. Pat. No. 4,263,400 on Apr. 21, 1981. The present invention is a result of further studies on the removal of the polysaccharide from the cultured material of the glucose isomerase producing microorganisms.